1Scientific RePoRtS | 7: 7437 | DOI:10.1038/s41598-017-07942-x www.nature.com/scientificreports Suppression of diabetic retinopathy with GLUT1 siRNA Zhi-Peng You, Yu-Lan Zhang, Ke Shi, Lu Shi, Yue-Zhi Zhang, Yue Zhou & Chang-yun Wang To investigate the effect of glucose transporter-1 (GLUT1) inhibition on diabetic retinopathy, we divided forty-eight mice into scrambled siRNA, diabetic scrambled siRNA, and GLUT1 siRNA (intravitreally injected) groups. Twenty-one weeks after diabetes induction, we calculated retinal glucose concentrations, used electroretinography (ERG) and histochemical methods to assess photoreceptor degeneration, and conducted immunoblotting, leukostasis and vascular leakage assays to estimate microangiopathy. The diabetic scrambled siRNA and GLUT1 siRNA exhibited higher glucose concentrations than scrambled siRNA, but GLUT1 siRNA group concentrations were only 50.05% of diabetic scrambled siRNA due to downregulated GLUT1 expression. The diabetic scrambled siRNA and GLUT1 siRNA had lower ERG amplitudes and ONL thicknesses than scrambled siRNA. However, compared with diabetic scrambled siRNA, GLUT1 siRNA group amplitudes and thicknesses were higher. Diabetic scrambled siRNA cones were more loosely arranged and had shorter outer segments than GLUT1 siRNA cones. ICAM-1 and TNF-α expression levels, adherent leukocyte numbers, fluorescence leakage areas and extravasated Evans blue in diabetic scrambled siRNA were higher than those in scrambled siRNA. However, these parameters in the GLUT1 siRNA were lower than diabetic scrambled siRNA. Together, these results demonstrate that GLUT1 siRNA restricted glucose transport by inhibiting GLUT1 expression, which decreased retinal glucose concentrations and ameliorated diabetic retinopathy. Diabetic retinopathy (DR) is one of the most common complications of diabetes mellitus (DM). DR often results in decreased vision and even blindness caused by macular edema, retinal detachment, and vitreous hemorrhage. The number of patients with diabetes may grow to 642 million in 20401. DR has been recognized as a microa- ngiopathy, as well as a neurodegenerative disease. Although the detailed mechanism underlying DR is unclear, two major global multicenter studies on diabetes, DCCT2 and UKPDS3, have revealed that a long-term high blood glucose level is the decisive factor for DR development. Moreover, excessive generation of retinal oxidative stress products4, activated protein kinase C5, and increased synthesis of glycosylated end products6 under the environment of high blood glucose levels initiate the impairment of retinal tissues and cells4. Since lesions are induced by high blood glucose levels, we hypothesize that DR progression can be relieved by restricting glucose transfer into the retina, thereby decreasing its local glucose content. Glucose transporter-1 (GLUT1) is the only currently known carrier of glucose through the blood–retinal barrier and is also respo ...

1. 1Scientific RePoRtS | 7: 7437 | DOI:10.1038/s41598-017-
07942-x
www.nature.com/scientificreports
Suppression of diabetic retinopathy
with GLUT1 siRNA
Zhi-Peng You, Yu-Lan Zhang, Ke Shi, Lu Shi, Yue-Zhi Zhang,
Yue Zhou & Chang-yun Wang
To investigate the effect of glucose transporter-1 (GLUT1)
inhibition on diabetic retinopathy,
we divided forty-eight mice into scrambled siRNA, diabetic
scrambled siRNA, and GLUT1 siRNA
(intravitreally injected) groups. Twenty-one weeks after
diabetes induction, we calculated retinal
glucose concentrations, used electroretinography (ERG) and
histochemical methods to assess
photoreceptor degeneration, and conducted immunoblotting,
leukostasis and vascular leakage assays
to estimate microangiopathy. The diabetic scrambled siRNA and
GLUT1 siRNA exhibited higher glucose
concentrations than scrambled siRNA, but GLUT1 siRNA group
concentrations were only 50.05% of
diabetic scrambled siRNA due to downregulated GLUT1
expression. The diabetic scrambled siRNA
and GLUT1 siRNA had lower ERG amplitudes and ONL
thicknesses than scrambled siRNA. However,
compared with diabetic scrambled siRNA, GLUT1 siRNA group
amplitudes and thicknesses were higher.
Diabetic scrambled siRNA cones were more loosely arranged
and had shorter outer segments than

2. GLUT1 siRNA cones. ICAM-1 and TNF-α expression levels,
adherent leukocyte numbers, fluorescence
leakage areas and extravasated Evans blue in diabetic scrambled
siRNA were higher than those in
scrambled siRNA. However, these parameters in the GLUT1
siRNA were lower than diabetic scrambled
siRNA. Together, these results demonstrate that GLUT1 siRNA
restricted glucose transport by
inhibiting GLUT1 expression, which decreased retinal glucose
concentrations and ameliorated diabetic
retinopathy.
Diabetic retinopathy (DR) is one of the most common
complications of diabetes mellitus (DM). DR often results
in decreased vision and even blindness caused by macular
edema, retinal detachment, and vitreous hemorrhage.
The number of patients with diabetes may grow to 642 million
in 20401. DR has been recognized as a microa-
ngiopathy, as well as a neurodegenerative disease. Although the
detailed mechanism underlying DR is unclear,
two major global multicenter studies on diabetes, DCCT2 and
UKPDS3, have revealed that a long-term high
blood glucose level is the decisive factor for DR development.
Moreover, excessive generation of retinal oxidative
stress products4, activated protein kinase C5, and increased
synthesis of glycosylated end products6 under the
environment of high blood glucose levels initiate the
impairment of retinal tissues and cells4. Since lesions are
induced by high blood glucose levels, we hypothesize that DR
progression can be relieved by restricting glucose
transfer into the retina, thereby decreasing its local glucose
content. Glucose transporter-1 (GLUT1) is the only
currently known carrier of glucose through the blood–retinal
barrier and is also responsible for the distribution
of glucose in ganglion cells, photoreceptor cells, and Müller
cells in the retina; GLUT1 is primarily expressed in

3. the vascular endothelial cells of the inner blood–retinal barrier
and retinal pigment epithelial cells of the outer
blood–retinal barrier7. GLUT1 was identified as a promising
target for diabetic retinopathy8, but current research
did not observe particular effect on retinopathy including
neuron degeneration and microangiopathy with means
of GLUT1 downregulation.
In this study, we intend to assess and compare
electroretinography (ERG) amplitudes, outer nuclear layer
(ONL) thicknesses, and cone cell densities in diabetic mice. The
results will be used to determine pathological
changes in photoreceptor cells, measure the expression levels of
retinal inflammatory factors, quantify adherent
leukocytes in retinal vessels and determine the leakage area of
the inner blood–retinal barrier to compare the level
of microangiopathy. Our purpose is to investigate the effect of
GLUT1 negative regulation on retinopathy via the
above parameters to verify whether suppression of GLUT1
would be benefit for DR.
Department of Ophthalmology, The Second Affiliated Hospital,
Nanchang University, Nanchang, 330006, China. Zhi-
Peng You and Yu-Lan Zhang contributed equally to this work.
Correspondence and requests for materials should be
addressed to K.S. (email: [email protected])
Received: 5 May 2017
Accepted: 5 July 2017
Published: xx xx xxxx
OPEN
mailto:[email protected]

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Results
Establishment of the diabetic model and measurement of body
weight and blood glucose lev-
els in the three groups. At 7 d after intraperitoneal injections
with streptozotocin, all blood glucose levels
of the 48 males C57BL/6 mice (diabetic scrambled siRNA and
GLUT1 siRNA groups) used for the establishment
of the diabetic model were greater than 300 mg/dL, and the
success rate of modeling was 100%. The body weight
and blood glucose levels of the mice were measured again at 20
weeks after the diabetic model was established.
No significant differences in the body weights of the mice were
found among the three groups when the diabetic
model was successfully established. However, the body weight
of the scrambled siRNA group was significantly
higher than that of the diabetic scrambled siRNA and GLUT1
siRNA groups by 40.44% and 35.59%, respectively,
at 20 weeks after the diabetic model was established (P < 0.01).
Both groups with diabetes exhibited an emaciated
body, whereas their water intake, food intake, and urine volume
were higher than those of the scrambled siRNA
group. At two time points: 1 d and 20 weeks after the diabetic
model establishment the blood glucose levels of the
scrambled siRNA group were lower than those of the diabetic
scrambled siRNA group by 46.85% and 55.37%,
respectively. The blood glucose levels were significantly lower
than those of the GLUT1 siRNA group by 47.36%
and 54.39% (P < 0.05). However, no significant difference was
found in the blood glucose levels between the dia-

5. betic scrambled siRNA and GLUT1 siRNA groups at both time
points (Table 1).
Determination of retinal glucose concentrations. The glucose
concentration in the retinal tissue of
the scrambled siRNA group was approximately 36.36 ± 2.98
nmol glucose/mg protein, whereas the glucose con-
centration in the retinal tissue of the diabetic scrambled siRNA
group increased to 156.73 ± 8.01 nmol glucose/
mg protein at 20 weeks after the diabetic model was established.
The glucose concentration in the GLUT1 siRNA
group was 78.44 ± 4.96 nmol glucose/mg protein. The glucose
concentrations in the retinal tissues of the diabetic
model mice of the two groups were significantly higher than
those in the mice of the scrambled siRNA group
(P < 0.01). However, the glucose concentration in the retinal
tissue of the GLUT1 siRNA group was significantly
lower than that in the diabetic scrambled siRNA group by
50.05% (P < 0.01) (Fig. 1a).
Retinal GLUT1 expression in the three groups. Immunoblotting
revealed that the expression of GLUT1
in the neural retinal layer was upregulated under diabetic
conditions, but the expression of retinal GLUT1 in the
GLUT1 siRNA group was lower than that in the scrambled
siRNA group by approximately 77.00%; however,
GLUT1 expression in the GLUT1 siRNA group was only lower
than that in the diabetic scrambled siRNA group
by 8.07%. Both of these differences were statistically
significant (P < 0.01) (Fig. 1b). Simultaneously, GLUT1
expression in the retinal pigment epithelium was also detected,
and the results were different from those obtained
in the neural retinal layer. Although GLUT1 expression in the
GLUT1 siRNA group was only 50.22% of that in the
diabetic scrambled siRNA group, which represented a
significant difference (P < 0.01), there was no significant

6. difference compared with that in the scrambled siRNA group (P
> 0.05) (Fig. 1c).
Pathological changes in cone photoreceptors. Photopic
electroretinogram amplitudes reflect the
function of cone photoreceptors9. The photopic ERG a and b
wave amplitudes of both the diabetic scrambled
siRNA and GLUT1 siRNA groups were significantly lower than
those of the scrambled siRNA group (P < 0.01).
However, the photopic ERG a and b wave amplitudes of the
GLUT1 siRNA group were significantly higher than
those of the diabetic scrambled siRNA group (Fig. 2a–c). Cone
photoreceptors were detected using an immuno-
fluorescence colocalization method. Compared with the
scrambled siRNA group, both the diabetic scrambled
siRNA and GLUT1 siRNA groups exhibited decreased cone cell
density and loosely arranged cones. The changes
were more significant in the diabetic scrambled siRNA group,
and the cone outer segments in the diabetic scram-
bled siRNA group appeared shorter than those in the GLUT1
siRNA treatment group (Fig. 2d–f ).
Pathological changes in rod cells. Scotopic ERG uses a gradient
of luminance to stimulate the retina
under dark conditions, which reflects rod cell function9. The
ERGs of the three groups are shown in Fig. 3a,b; the
scotopic ERG a and b wave amplitudes in both the diabetic
scrambled siRNA and GLUT1 siRNA groups were
significantly lower than those in the scrambled siRNA group (P
< 0.01). However, the scotopic ERG a and b wave
amplitudes in the GLUT1 siRNA group were significantly
higher than those in the diabetic scrambled siRNA
group. The ONL is primarily composed of photoreceptor nuclei,
and ONL thickness essentially reflects changes
in the number of rod photoreceptors because rods constitute
98% of all photoreceptors. In our study, ONL thick-

7. nesses were measured at 0.48, 0.96, 1.44, and 1.92 mm from the
optic nerve. At 20 weeks after the model was
established, the ONL thicknesses in the GLUT1 siRNA
treatment and diabetic scrambled siRNA groups were
group
body weight (g) blood glucose level (mg/dL)
at 1 d after
the diabetic
model was
established
at 20 weeks after
the diabetic model
was established
at 1 d after the
diabetic model
was established
at 20 weeks after the
diabetic model was
established
Scrambled siRNA 24.51 ± 2.13 34.21 ± 3.29 178.13 ± 24.31
173.41 ± 28.12
Diabetic scrambled
siRNA 23.12 ± 2.04 24.36 ± 3.23** 335.16 ± 63.37 388.54 ±
51.46**
GLUT1 siRNA
treatment 23.62 ± 2.12 25.23 ± 2.78** 338.42 ± 61.28 380.17 ±
65.81**

8. Table 1. Body weight and blood glucose levels of the three
groups (n = 16, x ± S). **P < 0.01, vs Scrambled
siRNA.
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lower than those in the scrambled siRNA group by
approximately 16.05% and 35.38%, respectively. However,
the ONL thicknesses in the GLUT1 siRNA treatment group were
significantly thicker than those in the diabetic
scrambled siRNA group by 29.92% (P < 0.05) (Fig. 3c–f ).
Inflammatory reactions in the retina. Previous research has
demonstrated that DR is an inflammatory
disease10, and ICAM-1 and TNF-α are two important
inflammation markers. Immunoblotting revealed that the
expression levels of ICAM-1 in the diabetic scrambled siRNA
and GLUT1 siRNA groups were significantly upreg-
ulated compared with those in the scrambled siRNA group (P <
0.01). However, the expression of retinal ICAM-1
in the GLUT1 siRNA group was approximately 66.14% of that
in the diabetic scrambled siRNA group (P < 0.05)
(Fig. 4a). Similar results were obtained for the expression levels
of TNF-α, which were also significantly upreg-
ulated in both the diabetic scrambled siRNA and GLUT1 siRNA
groups compared with those in the scrambled
siRNA group (P < 0.01). However, the expression of retinal
TNF-α in the GLUT1 siRNA group was approxi-
mately 54.76% of that in the diabetic scrambled siRNA group (P
< 0.05) (Fig. 4b).

9. Leukostasis is also an important indicator of retinal
inflammatory reactions9, as well as early pathological
changes in DR. No adherent leukocytes were found in the
scrambled siRNA group, whereas adherent leukocytes
were detected in the diabetic scrambled siRNA and GLUT1
siRNA groups. However, the number of adherent
leukocytes in the GLUT1 siRNA group was approximately
52.76% of that in the diabetic scrambled siRNA group
(P < 0.01) (Fig. 4a–d).
Blood–retinal barrier leakage. We used fluorescence microscopy
to observe and compare fluorescein
isothiocyanate-labeled bovine serum albumin as measurement of
inner blood–retinal barrier leakage. The results
showed that the inner blood–retinal barrier in the scrambled
siRNA group was intact, and no fluorescence leak-
age was observed, whereas fluorescence leakage regions were
detected in the diabetic scrambled siRNA and
GLUT1 siRNA groups. However, fewer fluorescence leakage
regions and smaller leakage areas were found in the
GLUT1 siRNA group than in the diabetic scrambled siRNA
group (Fig. 5a–c). We also used immunoblotting
to measure the content of retinal albumin, and the albumin
expression levels were also significantly increased
in both the diabetic scrambled siRNA and GLUT1 siRNA
groups compared with those in the scrambled siRNA
group (P < 0.01). However, the expression of retinal albumin in
the GLUT1 siRNA group was approximately
56.18% of that in the diabetic scrambled siRNA group (P <
0.01) (Fig. 5d). As shown in Fig. 5e, BRB permeability
was also measured in vivo using the Evans blue dye. The
concentration of Evans blue in formamide extract of
diabetic retina was significantly higher than scrambled siRNA
group (P < 0.01). GLUT1 siRNA treatment signif-
icantly reduced Evans blue extravasation compared to diabetic

10. scrambled siRNA group (P < 0.01).
Discussion
D R i s t h e o n e o f t h e m o s t c o m m o n a n d s e r i
o u s o c u l a r c o m p l i c a t i o n s , a n d i t s p a t h o -
g e n e s i s r e m a i n s u n c l e a r. T h e k e y e f f e c t s
o f h i g h b l o o d g l u c o s e l e v e l s i n D R a n d o t
h e r
d i a b e t e s - r e l a t e d c o m p l i c a t i o n s h a v e b e e
n d e m o n s t r a t e d i n t h e c l i n i c a l t r i a l s D C C
T 2 a n d
UKPDS3. The effects of high blood glucose on retinal cells may
include changes in the expression
Figure 1. (a) Determination of glucose concentration in retinal
tissues of the three groups, **P < 0.01 vs.
scrambled siRNA group, n = 6, x ± S. (b) GLUT1 expression in
the neural retinal layers of the three groups,
**P < 0.01 vs. scrambled siRNA group, ΔΔP < 0.01 vs. diabetic
scrambled siRNA group, n = 6, x ± S. (c) GLUT1
expression in the retinal pigment epithelia of the three groups,
**P < 0.01 vs. diabetic scrambled siRNA group,
ns: P > 0.05 vs. scrambled siRNA group, n = 6, x ± S. Full-
length blots are presented in Supplementary Figure 1
http://1
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levels of specific genes, buildup of advanced glycation end
products, and increased oxidative stress reactions11.
Given that the high-glucose microenvironment in DR damages

11. retinal tissues, controlling the glucose content of
local retinal tissues and reversing the high-glucose
microenvironment may address the problem. However, glu-
cose in the retina is transferred from blood circulation and
cannot pass through the phospholipid bilayer of mam-
malian cell membranes due to its water solubility; thus, GLUT,
a family of transport proteins, is used to transport
glucose12, which is required for retinal tissues to take up
glucose: GLUT1 is the only carrier for the transport of
glucose across the blood–retinal barrier7.
Researches concerning GLUT1 expression under high glucose
condition are contrary at present. Kumagai et
al. examined GLUT1 expression in the eyeballs (without or with
mild retinopathy) of patients with diabetes using
immunocytochemistry and found that the activity of retinal
GLUT1 in more than half of the eyeballs was 18 times
higher than that in the eyeballs of the normal control group13.
Fernandes et al. found that there was no compensa-
tory downregulation of GLUT1 on the inner BRB in diabetic
rats by means of immunogold staining14. However,
Fernandes et al. also reported that GLUT1 expression was
decreased in alloxan-induced diabetic rabbits15.
Similarly, Badr et al. suggested that diabetic condition
downregulated GLUT1 expression in the retina and its
microvessels16. These controversial results may attribute to
different animal species, diabetic course and meth-
odology. In our study, GLUT1 level in diabetic scrambled
siRNA group was 2.67 times that of scrambled siRNA
group.
Figure 2. (a) Representations of classic photopic ERG
waveforms. Figure 2b and c: Results of the statistical
analysis of photopic ERG a wave (b) and b wave (c) amplitudes
(n = 9) **P < 0.01, compared with scrambled
siRNA group, ΔΔP < 0.01, compared with diabetic scrambled

12. siRNA group. Figure 2d–f: Changes in cone
cells of the three groups were detected using an
immunofluorescence colocalization method (fluorescence
microscope ×400) (d) scrambled siRNA group, scale bar
represents 50 µm; (e) diabetic scrambled siRNA group;
(f) GLUT1 siRNA group; green: PNA, red: opsin, blue: DAPI;
OS: outer segment of cone cell; IS: inner segment
of cone cell.
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5Scientific RePoRtS | 7: 7437 | DOI:10.1038/s41598-017-
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siRNA is a type of RNA fragment that ranges in size from 19 bp
to 21 bp. siRNA can specifically degrade
mRNA of particular genes to inhibit the expression of these
genes. In our experiment, we administered effec-
tive GLUT1 siRNA sequences, which were identified in
previous studies17, to decrease the amount of glucose
transported into the retina. As mentioned above, no significant
difference in the overall blood glucose levels
was found between the mice with diabetes of both groups at 20
weeks after the diabetic model was established.
GLUT1 siRNA was intravitreally injected into mice of the
GLUT1 siRNA group, and the expression of retinal
GLUT1 was downregulated accordingly: it was decreased by
approximately 91.93% compared with that in the
diabetic scrambled siRNA group and by approximately 77%
compared with that in the scrambled siRNA group.
At the same time, the retinal glucose concentration in the
GLUT1 siRNA group was only 50.05% of that in the
diabetic scrambled siRNA group. This finding indicates that the
amount of glucose transported into the retina

13. was effectively reduced after GLUT1 was inhibited by GLUT1
siRNA. The retinal glucose concentration in the
GLUT1 siRNA group remained higher than that in the
scrambled siRNA group by approximately 53.64% because
intravitreal injections of GLUT1 siRNA significantly inhibited
GLUT1 within the inner blood–retinal barrier.
However, GLUT1 siRNA had a limited effect on the retinal
pigment epithelium, which forms the outer blood–ret-
inal barrier, and the expression of GLUT1 in the retinal pigment
epithelium was not downregulated. The biolog-
ical activities of GLUT1 have also been found to be upregulated
under diabetic conditions compared with those
under normal conditions18, 19. Consequently, the retinal
glucose transported from the outer blood–retinal barrier
resulted in higher retinal glucose concentrations in the GLUT1
siRNA group than those in the scrambled siRNA
group. Therefore, we established the conditions predicted in our
hypothesis by restricting GLUT1 in the inner
blood–retinal barrier. Next, we determined if the function and
morphology of photoreceptors and the level of
microangiopathy were affected using various indicators.
Figure 3. (a) Representations of classic scotopic ERG
waveforms. Figure 3b: Results of the statistical analysis
of scotopic ERG a wave and b wave amplitudes (n = 9) **P <
0.01, compared with scrambled siRNA group,
ΔΔP < 0.01, compared with diabetic scrambled siRNA group.
Figure 3c–f: ONL thicknesses of the three groups
(inverted microscope ×400) (d) scrambled siRNA group; (e)
diabetic scrambled siRNA group, scale bar
represents 50 μm; (f) GLUT1 siRNA group; (f) Statistical
analysis of ONL thicknesses.
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07942-x
Figure 4. Inflammatory reactions in the retina of mice in the
three groups. (a) Expression of retinal
inflammation marker ICAM-1 in mice of the three groups **P <
0.01 vs. scrambled siRNA group, ΔP < 0.05 vs.
diabetic scrambled siRNA group, n = 6, x ± S. (b) Expression
of retinal inflammation marker TNF-α in mice of
the three groups, **P < 0.01 vs. scrambled siRNA group, ΔΔP <
0.01 vs. diabetic scrambled siRNA group, n = 6,
x ± S, (c–f) Leukocytes adhesion to retinal vessel (c):
scrambled siRNA group, scale bar represents 100 µm
(upper row images)/scale bar represents 50 µm (lower row
images); (d) diabetic scrambled siRNA group; (e)
GLUT1 siRNA group; white arrows indicates adherent
leukocytes; (f) statistical analysis **P < 0.01 vs.
scrambled siRNA group, ΔΔP < 0.01 vs. diabetic scrambled
siRNA group, n = 6, x ± S (fluorescence microscope
×400). Full-length blots are presented in Supplementary
Figure 2.
Figure 5. Comparison of leakage of the inner blood–retinal
barrier among the three groups (a) scrambled
siRNA group; (b) diabetic scrambled siRNA group; (c) GLUT1
siRNA group, scale bar represents 200 µm,
fluorescence microscope ×400; white arrows indicate
fluorescence leakage regions; (d) Expression of retinal
albumin in mice of the three groups, (e) BRB permeability assay
using Evans blue dye in mice of the three
groups, **P < 0.01 vs. scrambled siRNA group, ΔΔP < 0.01 vs.
diabetic scrambled siRNA group, n = 6, x ± S.
Full-length blot is presented in Supplementary Figure 3.
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15. http://3
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Non-invasive recording is performed in ERG via platinum
electrodes at the surface of cornea, using flashes of
different brightness to stimulate the electrical activity of
photoreceptor cells9. Scotopic ERG and photopic ERG
are often used to measure the function of rod photoreceptors
and cone photoreceptors, respectively. Previous
studies have shown that abnormal ERGs are detected at the
early stage of diabetes in rats20. Another study has
reported that functional disorder of retinal photoreceptors
occurs in DR patients at the non-proliferative phase
before the onset of microangiopathic lesions, such as fundus
neovascularization, when inspected using flash
ERG21. In our study, although the scotopic ERG and photopic
ERG a and b wave amplitudes in the GLUT1 siRNA
group were lower than those in the scrambled siRNA group,
they were higher than those in the diabetic scram-
bled siRNA group. This finding indicates relatively mild
functional impairment in photoreceptor cells of mice
with diabetes after glucose transport into the retina was
restricted by GLUT1 siRNA.
ONL thickness was primarily used to measure rod
photoreceptors, and PNA was used to label cone photore-
ceptors. Another study has shown that the ONL thickness of
diabetic rats was gradually decreased over the course
of the illness20. Researchers have recognized that the
degeneration and death of rod cells are the primary cause of
abnormal visual function in patients with diabetes before the

16. presentation of DR and the associated important
pathological changes22. Moreover, in this study, the ONL
thicknesses in diabetic model mice of both groups were
lower than those in mice of the scrambled siRNA group at 20
weeks after the diabetic model was established. This
finding indicates that the rod cells in the diabetic model mice
were constantly dying throughout the experiment.
However, the ONL thickness at each time point in the GLUT1
siRNA group was higher than that in the diabetic
scrambled siRNA group. In addition to PNA labeling, we also
used S-opsin to mark the outer segments of cone
cells23 and found that the cone cells were more loosely
arranged and had shorter outer segments in the diabetic
scrambled siRNA group than those in the GLUT1 siRNA group,
as previously described. The above results sug-
gest that although photoreceptor cells were constantly dying
under diabetic conditions, the numbers of dead rod
and cone cells in the GLUT1 siRNA treatment group were
relatively low, which also demonstrates the protective
effect of a relatively low blood glucose microenvironment on
photoreceptor cells.
Inflammatory reactions are an important process in the
microangiopathy of DR; numerous studies have indi-
cated that the number of retinal leukocytes with enhanced
adhering ability and decreased deformability24, 25 is
increased in diabetic animal models. Adherent leukocytes
increase due to reduced passive deformability when
passing through capillary vessels with sizes less than the
diameter of the leukocytes in DR patients; adherent leu-
kocytes also significantly increase in number throughout the
progression of DR26. Therefore, a leukostasis assay
can be used for the analysis of inflammatory reaction levels in
DR. In our study, although the number of adherent
leukocytes in retinal vessels in the GLUT1 siRNA group was
higher than that in the scrambled siRNA group, it

17. was only 52.76% of the total number detected in the diabetic
scrambled siRNA group. We detected the expres-
sion levels of two inflammation markers simultaneously,
including chemotactic factor ICAM-1 and cytokine
TNF-α. ICAM-1 and its ligand CD18 play an important role in
mediating leukocyte adhesion27, and inhibition
of ICAM-1 results in significant mitigation of leukocyte
adhesion and vasopermeability28. Expression of TNF-α
is also upregulated in the retina under DR conditions29. The
expression levels of both inflammatory factors in the
retina of the GLUT1 siRNA group were only 66.14% and
54.76% of those in the diabetic scrambled siRNA group.
This finding indicates a relatively mild inflammatory reaction in
mice with diabetes after glucose transport into
the retina was restricted by GLUT1 siRNA. Damage to the
blood–retinal barrier is an important cause of retinal
edema, particularly macular edema, which might be ascribed to
the increase in leukostasis and upregulation of
inflammation marker expression9. As described above, the
numbers of adherent leukocytes and levels of inflam-
mation factors in the GLUT1 siRNA group were significantly
lower than those in the diabetic scrambled siRNA
group. When we examined the leakage of the inner blood–
retinal barrier, we identified fewer leakage regions and
smaller leakage areas in the GLUT1 siRNA group compared
with those in the diabetic scrambled siRNA group.
Similar result was obtained by Evans blue permeation assay.
These findings indicate that the relatively low blood
glucose microenvironment of the retina exerted a protective
effect on the inner blood–retinal barrier.
In summary, after an intravitreal injection of GLUT1 siRNA
was administered to inhibit GLUT1 in the retina,
the retinal glucose concentration in mice with diabetes was
decreased. Therefore, a retinal microenvironment
with relatively low glucose levels was formed. Under this

18. environment, pathological changes in the function
and morphology of retinal photoreceptors and the pathological
changes associated with microangiopathy were
relieved to some extent compared with those in mice with
diabetes, which suggests that restricting local retinal
glucose content by inhibiting GLUT1 might be a new direction
for the prevention and treatment of DR in the
future.
Materials and Methods
Synthesis of GLUT1 siRNA. An effective siRNA sequence was
designed according to reference17,
and Shanghai GenePharma Company synthesized the GLUT1-
targeted siRNA (positive-sense strand
5′-GGAATTCAATGCTGATGATGA-3′ and antisense strand 5′-
TCATCATCAGCATTGAATTCC-3′) and the
non-targeted siRNA as a negative control (positive-sense strand
5′-TTCTCCGAACGTGTCACGT-3′ and anti-
sense strand 5′-ACGTGACACGTTCGGAGAA-3′). Normal
saline treated with diethy pyrocarbonate (Sigma-
Aldrich Corp. St. Louis, MO, USA.) was used to dissolve
siRNA to reach a 20 μmol/L concentration.
Experimental animals and grouping. This study was carried out
in strict accordance with the recom-
mendations in the Guide for the Care and Use of Laboratory
Animals of the National Institutes of Health. The
protocol was approved by the Committee on the Ethics of
Animal Experiments of Nanchang University. All
surgeries were performed under ketamine & xylazine anesthesia,
and all efforts were made to minimize suffering.
A total of 48 male inbred line C57BL/6 mice at eight weeks of
age without eye diseases and weighing 20 g to 30 g
were purchased from the Animal Science Department, Nanchang
University. After we marked ear nails and serial
numbers for the mice, the animals were randomly divided into

19. scrambled siRNA, diabetic scrambled siRNA,
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8Scientific RePoRtS | 7: 7437 | DOI:10.1038/s41598-017-
07942-x
and GLUT1 siRNA treatment groups, with 16 mice in each
group. Establishment of DM model: Streptozotocin
(Sigma-Aldrich Corp. St. Louis, MO, USA.) was
intraperitoneally injected into mice for 5 consecutive days after
the mice fasted for 8 h. Streptozotocin (50 mg/kg body weight
in 0.01 mol/L citrate buffer solution [pH 4.5])
was intraperitoneally injected into the diabetic scrambled
siRNA and GLUT1 siRNA groups, whereas an equal
amount of citrate buffer solution was injected into the
scrambled siRNA group. A blood sample was collected
from the caudal vein to measure blood glucose levels at 7 d. The
standard for successful establishment of the DM
model was a blood glucose level > 300 mg/dL.
Intravitreal injection with siRNA. We performed intravitreal
injections in the first week after diabetes
induction. Intraperitoneal anesthesia with mixture of ketamine
and xylazine (Sigma-Aldrich Corp. St. Louis, MO,
USA.) was administered to the three groups and iodophor
disinfection was conducted around the eyes subse-
quently. A thirty-Gauge needle (Becton, Dickinson and
Company. Franklin Lakes, NJ, USA.) was inserted using
a Hamilton microinjector (Hamilton Company, Reno, NV,
U.S.A) toward the optic nerve at 1 mm outside of the
limbus under a microscope. The medicine was slowly injected
after the needle tip was detected in the pupil area.
A volume containing 1 μL of 20 μmol/L GLUT1 siRNA and 1

20. μL of transfection reagent was intravitreally injected
into the GLUT1 siRNA treatment group, whereas a volume
containing 1 μL of 20 μmol/L non-targeted siRNA
and 1 μL of transfection reagent (Invitrogen, Waltham, MA,
USA) was intravitreally injected into the scrambled
siRNA and diabetic scrambled siRNA groups. The injection was
conducted in both eyes and repeated twice a
week until nine injections were completed.
Electroretinography. Electroretinography was inspected at 20
weeks after the DM model was established.
All mice were dark-adapted overnight in a dark chamber after
pupil dilation was induced by tropicamide eye
drops (Santen Pharmaceutical Co., Ltd. Kita-ku, Osaka, Japan).
Anesthesia, consisting of ketamine and xyla-
zine, was administered the next day. The mice were then placed
on a heating board. The reference and ground
electrodes were inserted into the palate and tail, respectively.
Platinum corneal electrodes were placed on cornea
of both eyes, and recombinant bovine fibroblast growth factor
eye gel was applied for lubrication. Mouse ERG
preparation was completed under dim red lighting in the dark
chamber. Illumination intensities of 0.0004, 0.04,
4, 400, and 2000 cd•s/m2 were used to record scotopic ERG by
Espion electroretinogram E2 system (Diagnosys,
Lowell, MA, USA) Then, the mice were light adapted for 10
min, and photopic ERGs were recorded under an
illumination intensity of 2000 cd•s/m2.
Determination of retinal glucose concentrations. Six eyeballs
were enucleated for measurement of
retinal glucose concentrations. Retinal tissues were collected,
and 50 μL of deionized water was added to the
tissues. Samples were heated at 70 °C for 15 min, followed by
ultrasonication for 30 s, and centrifugation for
20 min. Up to 35 μL of supernatant was transferred into 165 μL

21. of reagent of a glucose concentration assay kit
(Sigma-Aldrich Corp. St. Louis, MO, USA.), followed by the
establishment of a standard curve and blank control.
A spectrum analyzer (SPECTRO Analytical Instruments GmbH,
Boschstr, Kleve, Germany) was used to measure
the optical density of the samples, and SPECTROstar Nano
MARS software (SPECTRO Analytical Instruments
GmbH, Boschstr, Kleve, Germany) was used to calculate
glucose concentrations. Subsequently, 10 μL of super-
natant was added to 190 μL of reagent of a protein
concentration assay (BIO-RAD Laboratories, Inc., Hercules,
CA, USA). A standard curve and blank control were also
established. The spectrum analyzer was used to measure
the optical density of the samples, and SPECTROstar Nano
MARS software was used to calculate protein con-
centrations. Retinal glucose concentration is presented as
nmol/mg, and the calculation formula was G × GV/
GMW × (P × PV), where G = glucose concentration (ng/mL),
GV = volume of liquid used to determine glucose
content (mL), P = protein concentration (mg/mL), PV = volume
of liquid used to determine protein content
(mL), and GMW = glucose molecular weight (180.2).
ONL thickness measurement. Eyeballs were enucleated and
directly fixed in 4% paraformaldehyde for
1 h. The cornea and lens were then removed, and the eyes were
fixed again in 4% paraformaldehyde for 15 min.
Subsequent steps were performed in accordance with a
conventional hematoxylin-eosin staining protocol. Slices
were sealed and observed under a microscope. ImagePro
software (Olympus Corporation, Tokyo, Japan) was
used to measure ONL thickness at 0.2, 0.4, 0.6, 0.8, 1.0, 1.2,
1.4, 1.6 and 1.8 mm from the optic nerve.
Immunofluorescence colocalization method. Eyeballs were
enucleated and directly fixed in 4% para-

22. formaldehyde for 1 h. The cornea and lens were then removed,
and the eyes were fixed again in 4% paraformal-
dehyde for 15 min. Subsequent steps were performed in
accordance with a conventional protocol. After paraffin
sections were prepared, dewaxing and heat-induced antigen
retrieval were performed in accordance with a con-
ventional protocol. The sections were then incubated with S-
opsin primary antibodies (Millipore Corporation. St.
Charles, MI, USA), followed by incubation with Peanut
agglutinin (PNA) (Vector Laboratories., Burlingame, CA,
USA) secondary antibodies the next day. After DAPI (Vector
Laboratories., Burlingame, CA, USA) was added,
the slices were observed under a fluorescence microscope.
Immunoblotting. Six eyeballs were enucleated, and retinal
tissues were collected and placed into Eppendorf
tubes with 200 μL of lysate. The remaining tissues- “eyecups”
were also mounted in tissue culture plate (Corning
Incorporated, Corning, NY, USA) and up to 5 μL of lysate was
added into the eyecups to extract retinal pigment
epithelial proteins. After 5 min, the lysates were collected.
Subsequent steps were performed in accordance with
a conventional protocol. Equal amounts of protein samples were
used for SDS-PAGE electrophoresis and trans-
membrane incubation with GLUT-1 (Millipore Corporation. St.
Charles, MI, USA), ICAM-1, TNF-α (Santa
Cruz Biotechnology, Inc., Dallas, TX, USA) and albumin
(Abcam plc, Cambridge, UK) primary antibodies. The
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9Scientific RePoRtS | 7: 7437 | DOI:10.1038/s41598-017-
07942-x

23. following day, secondary antibody incubation was conducted at
room temperature for 1 h after the membranes
were washed three times. Finally, the relative densities of the
blots were measured by UVP GelDoc-It Imager
(UVP LLC, Upland, CA, USA).
Leukostasis assay. Anesthesia, consisting of ketamine and
xylazine, was administered to three mice from
each of the three groups. The chest skin and ribs were cut open
to expose the thoracic cavity. The descending aorta
was closed by clamping, and the right auricle was cut open. A
27 G needle was inserted into left ventricle. Initially,
10 mL of PBS with heparin (0.1 mg/mL) was used to perfuse the
tissue and remove leukocytes that did not adhere
to retinal vessels. An additional volume of 20 μg/mL of PBS
and FITC- Concanavalin A (5 mg/kg) (Sigma-Aldrich
Corp. St. Louis, MO, USA.) was used to label adherent
leukocytes in retinal vessels. Up to 10 mL of PBS was
reused to remove excess FITC- Concanavalin A. The flow rate
of perfusion is 3–4 ml/min. Six eyeballs were enu-
cleated and directly fixed in 4% paraformaldehyde for 1 h.
Retinal flat mounts were prepared, and a fluorescence
microscope was used to observe and quantify the total number
of adherent leukocytes in the whole retina.
Blood–retinal barrier leakage. Ketamine and xylazine were used
to anesthetize three mice from each of
the three groups. FITC-BSA (66 kDa, 100 mg/kg) (Sigma-
Aldrich Corp. St. Louis, MO, USA.) was injected into
the femoral vein. The mice were killed after 20 min, and six
eyeballs from each group were enucleated and fixed
in 4% paraformaldehyde for 30 min. Retinal whole-mounts were
prepared, and blood–retinal barrier leakage was
observed under a fluorescence microscope.
Evans blue dye assay. Mouse was injected with received Evans

24. blue dye (45 mg/kg) (Sigma Aldrich, St.
Louis, MO, USA) via the tail vein. After 2 hours, 0.2 mL of
blood sample was drawn from re-anesthetized mice,
and mouse were perfused via the left ventricle with 200 mL PBS
to wash out dye. Retina was dissected out and
treated with dimethylformamide (Sigma Aldrich, St. Louis, MO,
USA) overnight at 70 °C for 18 hours. The extract
was centrifuged for 45 min. A spectrum analyzer (SPECTRO
Analytical Instruments GmbH, Boschstr, Kleve,
Germany) was used to test supernatant at 620 nm and 740 nm.
Blood samples were centrifuged for 15 min and
the supernatant was diluted 1:1000. The concentration of Evans
blue in the blood and retina was used to assess
BRB breakdown.
Statistical analysis. Statistical software SPSS17.0 was used to
perform analyses. The results are presented as
x ± S or x ± SEM, and chi-square test was used to compare
groups. P < 0.05 was considered significant.
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Acknowledgements
This work was supported by National Natural Science
Foundation of China (Grant number: 81460088), Jiangxi
Provincial Training Program for Distinguished Young Scholars
(Grant number: 20171BCB23092), Jiangxi
Provincial Key R&D Program (Grant number:
20171BBG70099), Jiangxi Provincial Natural Science
Foundation
for Youth Scientific Research (Grant number:
20171BAB215032), Scientific Research Foundation of Jiangxi
Education Department (Grant number: GJJ150270), Youth
Scientific Research Foundation of the Second
Affiliated Hospital of Nanchang University (Grant number:

29. 2014YNQN12011).
Author Contributions
Z.P.Y. and K.S. designed the study and performed the
experiments; Y.L.Z., L.S. and Y.Z.Z. performed the
experiments, Y.Z. and C.Y.W. analyzed the data, and K.S. wrote
the manuscript.
Additional Information
Supplementary information accompanies this paper at
doi:10.1038/s41598-017-07942-x
Competing Interests: The authors declare that they have no
competing interests.
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30. http://dx.doi.org/10.1038/s41598-017-07942-x
http://creativecommons.org/licenses/by/4.0/Suppression of
diabetic retinopathy with GLUT1 siRNA
Results
Establishment of the diabetic model and measurement of body
weight and blood glucose levels in the three groups.
Determination of retinal glucose concentrations. Retinal GLUT1
expression in the three groups. Pathological changes in cone
photoreceptors. Pathological changes in rod cells. Inflammatory
reactions in the retina. Blood–retinal barrier leakage.
Discussion
Materials and Methods
Synthesis of GLUT1 siRNA. Experimental animals and
grouping. Intravitreal injection with siRNA.
Electroretinography. Determination of retinal glucose
concentrations. ONL thickness measurement.
Immunofluorescence colocalization method. Immunoblotting.
Leukostasis assay. Blood–retinal barrier leakage. Evans blue
dye assay. Statistical analysis. Acknowledgements
Figure 1 (a) Determination of glucose concentration in retinal
tissues of the three groups, **P < 0.Figure 2 (a) Representations
of classic photopic ERG waveforms.Figure 3 (a)
Representations of classic scotopic ERG waveforms.Figure 4
Inflammatory reactions in the retina of mice in the three
groups.Figure 5 Comparison of leakage of the inner blood–
retinal barrier among the three groups (a) scrambled siRNA
group (b) diabetic scrambled siRNA group (c) GLUT1 siRNA
group, scale bar represents 200 µm, fluorescence microscope
×400 white arrows indicate flTable 1 Body weight and blood
glucose levels of the three groups (n = 16, ± S).